Microbial succession and dynamics in meromictic Mono Lake, California.

Mono Lake is a closed-basin, hypersaline, alkaline lake located in Eastern Sierra Nevada, California, that is dominated by microbial life. This unique ecosystem offers a natural laboratory for probing microbial community responses to environmental change. In 2017, a heavy snowpack and subsequent runoff led Mono Lake to transition from annually mixed (monomictic) to indefinitely stratified (meromictic). We followed microbial succession during this limnological shift, establishing a two-year (2017-2018) water-column time series of geochemical and microbiological data. Following meromictic conditions, anoxia persisted below the chemocline and reduced compounds such as sulfide and ammonium increased in concentration from near 0 to ~400 and ~150 µM, respectively, throughout 2018. We observed significant microbial succession, with trends varying by water depth. In the epilimnion (above the chemocline), aerobic heterotrophs were displaced by phototrophic genera when a large bloom of cyanobacteria appeared in fall 2018. Bacteria in the hypolimnion (below the chemocline) had a delayed, but systematic, response reflecting colonization by sediment "seed bank" communities. Phototrophic sulfide-oxidizing bacteria appeared first in summer 2017, followed by microbes associated with anaerobic fermentation in spring 2018, and eventually sulfate-reducing taxa by fall 2018. This slow shift indicated that multi-year meromixis was required to establish a sulfate-reducing community in Mono Lake, although sulfide oxidizers thrive throughout mixing regimes. The abundant green alga Picocystis remained the dominant primary producer during the meromixis event, abundant throughout the water column including in the hypolimnion despite the absence of light and prevalence of sulfide. Our study adds to the growing literature describing microbial resistance and resilience during lake mixing events related to climatic events and environmental change.

Arsenolipids in Cultured Picocystis Strain ML and Their Occurrence in Biota and Sediment from Mono Lake, California.

Primary production in Mono Lake, a hypersaline soda lake rich in dissolved inorganic arsenic, is dominated by Picocystis strain ML. We set out to determine if this photoautotrophic picoplankter could metabolize inorganic arsenic and in doing so form unusual arsenolipids (e.g., arsenic bound to 2-O-methyl ribosides) as reported in other saline ecosystems and by halophilic algae. We cultivated Picocystis strain ML on a seawater-based medium with either low (37 µM) or high (1000 µM) phosphate in the presence of arsenite (400 µM), arsenate (800 µM), or without arsenic additions (ca 0.025 µM). Cultivars formed a variety of organoarsenic compounds, including a phytyl 2-O-methyl arsenosugar, depending upon the cultivation conditions and arsenic exposure. When the cells were grown at low P, the organoarsenicals they produced when exposed to both arsenite and arsenate were primarily arsenolipids (~88%) with only a modest content of water-soluble organoarsenic compounds (e.g., arsenosugars). When grown at high P, sequestration shifted to primarily water-soluble, simple methylated arsenicals such as dimethylarsinate; arsenolipids still constituted ~32% of organoarsenic incorporated into cells exposed to arsenate but < 1% when exposed to arsenite. Curiously, Picocystis strain ML grown at low P and exposed to arsenate sequestered huge amounts of arsenic into the cells accounting for 13.3% of the dry biomass; cells grown at low P and arsenite exposure sequestered much lower amounts, equivalent to 0.35% of dry biomass. Extraction of a resistant phase with trifluoroacetate recovered most of the sequestered arsenic in the form of arsenate. Uptake of arsenate into low P-cultivated cells was confirmed by X-ray fluorescence, while XANES/EXAFS spectra indicated the sequestered arsenic was retained as an inorganic iron precipitate, similar to scorodite, rather than as an As-containing macromolecule. Samples from Mono Lake demonstrated the presence of a wide variety of organoarsenic compounds, including arsenosugar phospholipids, most prevalent in zooplankton (Artemia) and phytoplankton samples, with much lower amounts detected in the bottom sediments. These observations suggest a trophic transfer of organoarsenicals from the phytoplankton (Picocystis) to the zooplankton (Artemia) community, with efficient bacterial mineralization of any lysis-released organoarsenicals back to inorganic oxyanions before they sink to the sediments.

Metabolic Capability and Phylogenetic Diversity of Mono Lake during a Bloom of the Eukaryotic Phototroph Picocystis sp. Strain ML.

Algal blooms in lakes are often associated with anthropogenic eutrophication; however, they can occur without the human introduction of nutrients to a lake. A rare bloom of the alga Picocystis sp. strain ML occurred in the spring of 2016 at Mono Lake, a hyperalkaline lake in California, which was also at the apex of a multiyear-long drought. These conditions presented a unique sampling opportunity to investigate microbiological dynamics and potential metabolic function during an intense natural algal bloom. We conducted a comprehensive molecular analysis along a depth transect near the center of the lake from the surface to a depth of 25 m in June 2016. Across sampled depths, rRNA gene sequencing revealed that Picocystis-associated chloroplasts were found at 40 to 50% relative abundance, greater than values recorded previously. Despite high relative abundances of the photosynthetic oxygenic algal genus Picocystis, oxygen declined below detectable limits below a depth of 15 m, corresponding with an increase in microorganisms known to be anaerobic. In contrast to previously sampled years, both metagenomic and metatranscriptomic data suggested a depletion of anaerobic sulfate-reducing microorganisms throughout the lake's water column. Transcripts associated with photosystem I and II were expressed at both 2 m and 25 m, suggesting that limited oxygen production could occur at extremely low light levels at depth within the lake. Blooms of Picocystis appear to correspond with a loss of microbial activity such as sulfate reduction within Mono Lake, yet microorganisms may survive within the sediment to repopulate the lake water column as the bloom subsides.IMPORTANCE Mono Lake, California, provides a habitat to a unique ecological community that is heavily stressed due to recent human water diversions and a period of extended drought. To date, no baseline information exists from Mono Lake to understand how the microbial community responds to human-influenced drought or algal bloom or what metabolisms are lost in the water column as a consequence of such environmental pressures. While previously identified anaerobic members of the microbial community disappear from the water column during drought and bloom, sediment samples suggest that these microorganisms survive at the lake bottom or in the subsurface. Thus, the sediments may represent a type of seed bank that could restore the microbial community as a bloom subsides. Our work sheds light on the potential photosynthetic activity of the halotolerant alga Picocystis sp. strain ML and how the function and activity of the remainder of the microbial community responds during a bloom at Mono Lake.

Acetylenotrophy: a hidden but ubiquitous microbial metabolism?

Acetylene (IUPAC name: ethyne) is a colorless, gaseous hydrocarbon, composed of two triple bonded carbon atoms attached to hydrogens (C2H2). When microbiologists and biogeochemists think of acetylene, they immediately think of its use as an inhibitory compound of certain microbial processes and a tracer for nitrogen fixation. However, what is less widely known is that anaerobic and aerobic microorganisms can degrade acetylene, using it as a sole carbon and energy source and providing the basis of a microbial food web. Here, we review what is known about acetylene degrading organisms and introduce the term 'acetylenotrophs' to refer to the microorganisms that carry out this metabolic pathway. In addition, we review the known environmental sources of acetylene and postulate the presence of an hidden acetylene cycle. The abundance of bacteria capable of using acetylene and other alkynes as an energy and carbon source suggests that there are energy cycles present in the environment that are driven by acetylene and alkyne production and consumption that are isolated from atmospheric exchange. Acetylenotrophs may have developed to leverage the relatively high concentrations of acetylene in the pre-Cambrian atmosphere, evolving later to survive in specialized niches where acetylene and other alkynes were produced.

Stable Carbon Isotope Fractionation during Bacterial Acetylene Fermentation: Potential for Life Detection in Hydrocarbon-Rich Volatiles of Icy Planet(oid)s.

UNLABELLED: We report the first study of stable carbon isotope fractionation during microbial fermentation of acetylene (C2H2) in sediments, sediment enrichments, and bacterial cultures. Kinetic isotope effects (KIEs) averaged 3.7 ± 0.5‰ for slurries prepared with sediment collected at an intertidal mudflat in San Francisco Bay and 2.7 ± 0.2‰ for a pure culture of Pelobacter sp. isolated from these sediments. A similar KIE of 1.8 ± 0.7‰ was obtained for methanogenic enrichments derived from sediment collected at freshwater Searsville Lake, California. However, C2H2 uptake by a highly enriched mixed culture (strain SV7) obtained from Searsville Lake sediments resulted in a larger KIE of 9.0 ± 0.7‰. These are modest KIEs when compared with fractionation observed during oxidation of C1 compounds such as methane and methyl halides but are comparable to results obtained with other C2 compounds. These observations may be useful in distinguishing biologically active processes operating at distant locales in the Solar System where C2H2 is present. These locales include the surface of Saturn's largest moon Titan and the vaporous water- and hydrocarbon-rich jets emanating from Enceladus. KEY WORDS: Acetylene-Fermentation-Isotope fractionation-Enceladus-Life detection.

Microbiological oxidation of antimony(III) with oxygen or nitrate by bacteria isolated from contaminated mine sediments.

Bacterial oxidation of arsenite [As(III)] is a well-studied and important biogeochemical pathway that directly influences the mobility and toxicity of arsenic in the environment. In contrast, little is known about microbiological oxidation of the chemically similar anion antimonite [Sb(III)]. In this study, two bacterial strains, designated IDSBO-1 and IDSBO-4, which grow on tartrate compounds and oxidize Sb(III) using either oxygen or nitrate, respectively, as a terminal electron acceptor, were isolated from contaminated mine sediments. Both isolates belonged to the Comamonadaceae family and were 99% similar to previously described species. We identify these novel strains as Hydrogenophaga taeniospiralis strain IDSBO-1 and Variovorax paradoxus strain IDSBO-4. Both strains possess a gene with homology to the aioA gene, which encodes an As(III)-oxidase, and both oxidize As(III) aerobically, but only IDSBO-4 oxidized Sb(III) in the presence of air, while strain IDSBO-1 could achieve this via nitrate respiration. Our results suggest that expression of aioA is not induced by Sb(III) but may be involved in Sb(III) oxidation along with an Sb(III)-specific pathway. Phylogenetic analysis of proteins encoded by the aioA genes revealed a close sequence similarity (90%) among the two isolates and other known As(III)-oxidizing bacteria, particularly Acidovorax sp. strain NO1. Both isolates were capable of chemolithoautotrophic growth using As(III) as a primary electron donor, and strain IDSBO-4 exhibited incorporation of radiolabeled [(14)C]bicarbonate while oxidizing Sb(III) from Sb(III)-tartrate, suggesting possible Sb(III)-dependent autotrophy. Enrichment cultures produced the Sb(V) oxide mineral mopungite and lesser amounts of Sb(III)-bearing senarmontite as precipitates.

Methane Oxidation and Molecular Characterization of Methanotrophs from a Former Mercury Mine Impoundment.

The Herman Pit, once a mercury mine, is an impoundment located in an active geothermal area. Its acidic waters are permeated by hundreds of gas seeps. One seep was sampled and found to be composed of mostly CO2 with some CH4 present. The δ(13)CH4 value suggested a complex origin for the methane: i.e., a thermogenic component plus a biological methanogenic portion. The relatively (12)C-enriched CO2 suggested a reworking of the ebullitive methane by methanotrophic bacteria. Therefore, we tested bottom sediments for their ability to consume methane by conducting aerobic incubations of slurried materials. Methane was removed from the headspace of live slurries, and subsequent additions of methane resulted in faster removal rates. This activity could be transferred to an artificial, acidic medium, indicating the presence of acidophilic or acid-tolerant methanotrophs, the latter reinforced by the observation of maximum activity at pH = 4.5 with incubated slurries. A successful extraction of sterol and hopanoid lipids characteristic of methanotrophs was achieved, and their abundances greatly increased with increased sediment methane consumption. DNA extracted from methane-oxidizing enrichment cultures was amplified and sequenced for pmoA genes that aligned with methanotrophic members of the Gammaproteobacteria. An enrichment culture was established that grew in an acidic (pH 4.5) medium via methane oxidation.

Methane oxidation linked to chlorite dismutation.

We examined the potential for CH4 oxidation to be coupled with oxygen derived from the dissimilatory reduction of perchlorate, chlorate, or via chlorite (ClO(-) 2) dismutation. Although dissimilatory reduction of ClO(-) 4 and ClO(-) 3 could be inferred from the accumulation of chloride ions either in spent media or in soil slurries prepared from exposed freshwater lake sediment, neither of these oxyanions evoked methane oxidation when added to either anaerobic mixed cultures or soil enriched in methanotrophs. In contrast, ClO(-) 2 amendment elicited such activity. Methane (0.2 kPa) was completely removed within several days from the headspace of cell suspensions of Dechloromonas agitata CKB incubated with either Methylococcus capsulatus Bath or Methylomicrobium album BG8 in the presence of 5 mM ClO(-) 2. We also observed complete removal of 0.2 kPa CH4 in bottles containing soil enriched in methanotrophs when co-incubated with D. agitata CKB and 10 mM ClO(-) 2. However, to be effective these experiments required physical separation of soil from D. agitata CKB to allow for the partitioning of O2 liberated from chlorite dismutation into the shared headspace. Although a link between ClO(-) 2 and CH4 consumption was established in soils and cultures, no upstream connection with either ClO(-) 4 or ClO(-) 3 was discerned. This result suggests that the release of O2 during enzymatic perchlorate reduction was negligible, and that the oxygen produced was unavailable to the aerobic methanotrophs.

Microbiological reduction of Sb(V) in anoxic freshwater sediments.

Microbiological reduction of millimolar concentrations of Sb(V) to Sb(III) was observed in anoxic sediments from two freshwater settings: (1) a Sb- and As-contaminated mine site (Stibnite Mine) in central Idaho and 2) an uncontaminated suburban lake (Searsville Lake) in the San Francisco Bay Area. Rates of Sb(V) reduction in anoxic sediment microcosms and enrichment cultures were enhanced by amendment with lactate or acetate as electron donors but not by H2, and no reduction occurred in sterilized controls. Addition of 2-(14)C-acetate to Stibnite Mine microcosms resulted in the production of (14)CO2 coupled to Sb(V) reduction, suggesting that this process proceeds by a dissimilatory respiratory pathway in those sediments. Antimony(V) reduction in Searsville Lake sediments was not coupled to acetate mineralization and may be associated with Sb-resistance. The microcosms and enrichment cultures also reduced sulfate, and the precipitation of insoluble Sb(III)-sulfide complexes was a major sink for reduced Sb. The reduction of Sb(V) by Stibnite Mine sediments was inhibited by As(V), suggesting that As(V) is a preferred electron acceptor for the indigenous community. These findings indicate a novel pathway for anaerobic microbiological respiration and suggest that communities capable of reducing high concentrations of Sb(V) commonly occur naturally in the environment.

Desulfohalophilus alkaliarsenatis gen. nov., sp. nov., an extremely halophilic sulfate- and arsenate-respiring bacterium from Searles Lake, California.

A haloalkaliphilic sulfate-respiring bacterium, strain SLSR-1, was isolated from a lactate-fed stable enrichment culture originally obtained from the extreme environment of Searles Lake, California. The isolate proved capable of growth via sulfate-reduction over a broad range of salinities (125-330 g/L), although growth was slowest at salt-saturation. Strain SLSR-1 was also capable of growth via dissimilatory arsenate-reduction and displayed an even broader range of salinity tolerance (50-330 g/L) when grown under these conditions. Strain SLSR-1 could also grow via dissimilatory nitrate reduction to ammonia. Growth experiments in the presence of high borate concentrations indicated a greater sensitivity of sulfate-reduction than arsenate-respiration to this naturally abundant anion in Searles Lake. Strain SLSR-1 contained genes involved in both sulfate-reduction (dsrAB) and arsenate respiration (arrA). Amplicons of 16S rRNA gene sequences obtained from DNA extracted from Searles Lake sediment revealed the presence of close relatives of strain SLSR-1 as part of the flora of this ecosystem despite the fact that sulfate-reduction activity could not be detected in situ. We conclude that strain SLSR-1 can only achieve growth via arsenate-reduction under the current chemical conditions prevalent at Searles Lake. Strain SLSR-1 is a deltaproteobacterium in the family Desulfohalobiacea of anaerobic, haloalkaliphilic bacteria, for which we propose the name Desulfohalophilus alkaliarsenatis gen. nov., sp. nov.

Electricity generation by anaerobic bacteria and anoxic sediments from hypersaline soda lakes.

Anaerobic bacteria and anoxic sediments from soda lakes produced electricity in microbial fuel cells (MFCs). No electricity was generated in the absence of bacterial metabolism. Arsenate respiring bacteria isolated from moderately hypersaline Mono Lake (Bacillus selenitireducens), and salt-saturated Searles Lake, CA (strain SLAS-1) oxidized lactate using arsenate as the electron acceptor. However, these cultures grew equally well without added arsenate using the MFC anode as their electron acceptor, and in the process oxidized lactate more efficiently. The decrease in electricity generation by consumption of added alternative electron acceptors (i.e. arsenate) which competed with the anode for available electrons proved to be a useful indicator of microbial activity and hence life in the fuel cells. Shaken sediment slurries from these two lakes also generated electricity, with or without added lactate. Hydrogen added to sediment slurries was consumed but did not stimulate electricity production. Finally, electricity was generated in statically incubated "intact" sediment cores from these lakes. More power was produced in sediment from Mono Lake than from Searles Lake, however microbial fuel cells could detect low levels of metabolism operating under moderate and extreme conditions of salt stress.

Aminobacter ciceronei sp. nov. and Aminobacter lissarensis sp. nov., isolated from various terrestrial environments.

The bacterial strains IMB-1(T) and CC495(T), which are capable of growth on methyl chloride (CH(3)Cl, chloromethane) and methyl bromide (CH(3)Br, bromomethane), were isolated from agricultural soil in California fumigated with CH(3)Br, and woodland soil in Northern Ireland, respectively. Two pesticide-/herbicide-degrading bacteria, strains ER2 and C147, were isolated from agricultural soil in Canada. Strain ER2 degrades N-methyl carbamate insecticides, and strain C147 degrades triazine herbicides widely used in agriculture. On the basis of their morphological, physiological and genotypic characteristics, these four strains are considered to represent two novel species of the genus Aminobacter, for which the names Aminobacter ciceronei sp. nov. (type strain IMB-1(T)=ATCC 202197(T)=CIP 108660(T)=CCUG 50580(T); strains ER2 and C147) and Aminobacter lissarensis sp. nov. (type strain CC495(T)=NCIMB 13798(T)=CIP 108661(T)=CCUG 50579(T)) are proposed.

A microbial arsenic cycle in a salt-saturated, extreme environment.

Searles Lake is a salt-saturated, alkaline brine unusually rich in the toxic element arsenic. Arsenic speciation changed from arsenate [As(V)] to arsenite [As(III)] with sediment depth. Incubated anoxic sediment slurries displayed dissimilatory As(V)-reductase activity that was markedly stimulated by H2 or sulfide, whereas aerobic slurries had rapid As(III)-oxidase activity. An anaerobic, extremely haloalkaliphilic bacterium was isolated from the sediment that grew via As(V) respiration, using either lactate or sulfide as its electron donor. Hence, a full biogeochemical cycle of arsenic occurs in Searles Lake, driven in part by inorganic electron donors.

Bioreactors for removing methyl bromide following contained fumigations.

Use of methyl bromide (MeBr) as a quarantine, commodity, or structural fumigant is under scrutiny because its release to the atmosphere contributes to the depletion of stratospheric ozone. A closed-system bioreactor consisting of 0.5 L of a growing culture of a previously described bacterium, strain IMB-1, removed MeBr (> 110 micromol L(-1)) from recirculating air. Strain IMB-1 grew slowly to high cell densities in the bioreactor using MeBr as its sole carbon and energy source. Bacterial oxidation of MeBr produced CO2 and hydrobromic acid (HBr), which required continuous neutralization with NaOH for the system to operate effectively. Strain IMB-1 was capable of sustained oxidation of large amounts of MeBr (170 mmol in 46 d). In an open-system bioreactor (10-L fermenter), strain IMB-1 oxidized a continuous supply of MeBr (220 /micromol L(-1) in air). Growth was continuous, and 0.5 mol of MeBr was removed from the air supply in 14 d. The specific rate of MeBr oxidation was 7 x 10(-16) mol cell(-1) h(-1). Bioreactors such as these can therefore be used to remove large quantities of contaminant MeBr, which opens the possibility of biodegradation as a practical means for its disposal.